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Studies on the protective effect of dietary fish oil on cisplatin inducednephrotoxicity in rats

Ashreeb Naqshbandi, Md. Wasim Khan, Sana Rizwan, Sayeed ur Rehman, Farah Khan ⇑Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, UP, India

a r t i c l e i n f o

Article history:Received 7 July 2011Accepted 5 October 2011Available online 14 October 2011

Keywords:CisplatinNephrotoxicityFish oilBrush border enzymesCarbohydrate metabolismOxidative stress

a b s t r a c t

Cisplatin (CP) is a major antineoplastic drug for the treatment of solid tumors, however, dose dependentnephrotoxicity remains the major concern for its long term use. Several agents/strategies were attemptedto prevent CP nephrotoxicity but were not found suitable for clinical practice. Dietary fish oil (FO)enriched in x-3 fatty acids has been shown to prevent/reduce the progression of certain types of cancers,cardiovascular and renal disorders. The present study was undertaken to see whether FO can prevent CP-induced nephrotoxic and other deleterious effects. Rats were prefed experimental diets for 10 days andthen received a single dose of CP (6 mg/kg body weight) intraperitoneally while still on diet. Serum/urineparameters, enzymes of carbohydrate metabolism, brush border membrane (BBM) and oxidative stress inrat kidney were analyzed. CP nephrotoxicity was recorded by increased serum creatinine and blood ureanitrogen. CP decreased the activities of metabolic enzymes, antioxidant defense system and BBMenzymes. In contrast, FO alone increased enzyme activities of carbohydrate metabolism and brush bordermembrane (BBM). FO feeding to CP treated rats markedly enhanced resistance to CP-elicited deleteriouseffects. Dietary FO supplementation ameliorated CP induced specific metabolic alterations and oxidativedamage due to its intrinsic biochemical antioxidant properties.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Cisplatin (cis-diamminedichloroplatinum II, CP) one of the mosteffective chemotherapeutic agent that plays a major role in thetreatment of variety of human solid tumors including those ofthe head, neck, testis, ovary and breast (Lebwohl and Canetta,1998). Inspite of its beneficial antitumor action, dose related neph-rotoxicity and neurotoxicity limits its application in clinical oncol-ogy (Antonio et al., 2002).

Light and electron microscopy have shown that the CP-inducedinjury and necrosis in the rat kidney are predominantly localized inS3 subsegments of proximal tubular epithelial cells (Townsendet al., 2003). Morphologically, it is characterized by the loss of

microvilli, cellular swelling, and condensation of nuclear chroma-tin (Kuhlmann et al., 1997). Mitochondria, lysosomes and micro-somes are critical CP targets (Zhang and Lindup, 1993;Leibbrandt et al., 1995). Functional alterations are characterizedby change in urine volume, increase in blood urea nitrogen and ser-um creatinine (Yao et al., 2007).

The mechanism underlying the side effects induced by cisplatinis not understood clearly, however it was considered to be attrib-uted to the combination of multi-ways (Hong et al., 2005; Rameshand Reeves, 2002; Nowak, 2002; Townsend and Hanigan, 2002;Xiao et al., 2003) such as the generation of reactive oxygen species(ROS), which could interfere with the antioxidant defense systemand result in oxidative damage in different tissues (Kuhlmannet al., 1997;Matsushima et al., 1998; Kuhad et al., 2007; Khanet al., 2009a,b) and reaction with thiols in protein and glutathione,which could cause cell dysfunction.

Various studies have focused on the ways for prevention of cis-platin associated side effects via supplementation of preventiveagents simultaneously. These include antioxidants, modulators ofnitric oxide, diuretics, and cytoprotective and apoptotic agents(Ali and Al Moundhri, 2006). However, none of these were foundto be suitable.

Renewed interest has been observed in recent years in x-3 fattyacids enriched fish oil (FO) diet that has profound beneficial healtheffects against various pathologies (Simopoulos, 1991) including

0278-6915/$ - see front matter � 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.fct.2011.10.039

Abbreviations: ACPase, acid phosphatase; ALP, alkaline phosphatase; ALT,alanine aminotranferase; AST, aspartate aminotransferase; BBM, brush bordermembrane; BBMV, brush border membrane vesicles; BUN, blood urea nitrogen; CP,cisplatin; CPF, cisplatin-fish oil; FO, fish oil; FBPase, fructose-1,6-bisphosphatase;G6Pase, glucose-6-phosphatase; G6PDH, glucose-6-phosphate dehydrogenase;GGTase, c-glutamyl transferase; GSH-Px, glutathione peroxidase; HK, hexokinase;LAP, leucine aminopeptidase; LDH, lactate dehydrogenase; MDH, malate dehydro-genase; ME, malic enzyme; NADP+, nicotinamide adenine dinucleotide phosphate;PUFA, polyunsaturated fatty acids; ROS, reactive oxygen species; Scr, serumcreatinine; SOD, superoxide dismutase.⇑ Corresponding author. Tel.: +91 571 2700741; fax: +91 571 2706002.

E-mail address: drfarah23@live.com (F. Khan).

Food and Chemical Toxicology 50 (2012) 265–273

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cardiovascular, respiratory, inflammatory and immune renal dis-eases; diabetes, depression and cancer (Caterina et al., 1994;Aronson et al., 2001; Donadio, 2001; Hibbeln, 1998; Posta et al.,2006). Previous studies have shown protective role of dietary FOagainst drug induced nephrotoxicity (Abdel-Gayoum et al., 1995;Thakkar et al., 2000). However, it is important that the prospectivenephro protective agent should not affect the drug efficacy whilereducing its side effects. There is evidence that dietary supplemen-tation of FO enhances the antitumor effects of CP chemotherapy(Yam et al., 2001). Recent study from our lab has shown that die-tary FO supplementation ameliorates GM induced nephrotoxic ef-fects (Priyamvada et al., 2008). However, the protective effect of FOon CP induced nephrotoxicity remains uninvestigated.

Considering the potential clinical use of CP and numeroushealth benefits of FO, the present work was undertaken to studythe biochemical events/cellular response/mechanisms of CPnephropathy and its protection by dietary FO. We hypothesizedthat fish oil would prevent CP-induced nephrotoxicity due to itsintrinsic biochemical and antioxidant properties that would leadto improved metabolism and antioxidant defense mechanism ofthe kidney. The results obtained indicate that dietary supplemen-tation with fish oil markedly ameliorate CP induced nephrotoxicityparameters and support a potential therapeutic use of CP + FOcombination in combating cancer without nephrotoxic and otherharmful side effects.

2. Materials and methods

2.1. Chemicals and drugs

Fish oil of menhaden and cisplatin from Sigma Chemical Co., (St. Louis, MO,USA). All other chemicals used were of analytical grade and were purchased eitherfrom Sigma Chemical Co. or SRL (Mumbai, India).

2.2. Diet

A nutritionally adequate laboratory pellet diet was obtained from AashirwaadIndustries, Chandigarh (India). Pellets were crushed finely and mixed with 15% fishoil and stored in airtight containers. Vitamin E as DL-a-tocopherol (270 mg/kg chow)was added to each of the modified rat chows in order to meet the increased meta-bolic requirement for antioxidants on a diet high in polyunsaturated fatty acids.

2.3. Experimental design

The animal experiments were conducted according to the guidelines of Com-mittee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA),Ministry of Environment and Forests, Government of India. Adult male Wistar rats(8 rats/group) weighing between 150 and 200 g were used in the study. Animalswere acclimatized to the animal facility for a week on standard rat chow and al-lowed water ad libitum under controlled conditions of 25 ± 2 �C temperature,

50 ± 15% relative humidity and normal photoperiod (12 h dark and light). Fourgroups of rats entered the study after acclimatization (Fig. 1). They were fed oneither normal diet (control and CP groups) or diet containing 15% fish oil (CPFand FO groups). After 10 days rats in two groups (CP and CPF) were administereda single dose of CP intraperitoneally (6 mg/kg body weight) in 0.9% saline. Animalsin the control and FO group received an equivalent amount of normal saline. Therats were sacrificed 4 days after CP administration under light ether anesthesia.Blood and urine samples were collected and kidneys were removed and processedfor the preparation of homogenates and BBMV (brush border membrane vesicles) asdescribed below. All the preparations and analyses of various parameters were per-formed simultaneously under similar experimental conditions to avoid any day today variations. Body weight of rats were recorded at the start and completion ofthe experimental procedures.

2.4. Preparation of homogenates

After the completion of the experiment, the kidneys were removed, decapsulat-ed and kept in ice-cold buffered saline (154 mM NaCl, 5 mM Tris–HEPES, pH 7.5).The cortex was carefully separated from medulla as described earlier (Khundmiriet al., 2004). A 15% (w/v) homogenate was prepared in 0.1 M Tris–HCl buffer, pH7.5, using Potter–Elvehejem homogenizer (Remi Motors, Mumbai, India); by pass-ing five pulses. The homogenate was centrifuged at 3000g at 4 �C for 15 min to re-move cell debris and the supernatant was saved in aliquots and stored at �20 �C forassaying the enzymes of carbohydrate metabolism, free-radical scavenging en-zymes and for estimation of total-SH and lipid peroxidation (LPO).

2.5. Preparation of brush border membrane

BBMV were prepared from whole cortex using the MgCl2 precipitation methodas described previously (Khundmiri et al., 2005). Briefly, freshly minced corticalslices were homogenized in 50 mM mannitol and 5 mM Tris–HEPES buffer, pH7.0 (20 ml of buffer/g of cortical tissue), in a glass Teflon homogenizer with fourcomplete strokes. The homogenate was then subjected to high speed Ultra-Turrexhomogenizer (Type T-25, Janke & Kunkel GMBH & Co., KG. Staufen) for three strokesof 15 s each with an interval of 15 s between each stroke. MgCl2 was added to thehomogenate to a final concentration of 10 mM and the mixture stirred for 20 minon ice. The homogenate was centrifuged at 2000g for 10 min in a Beckman centri-fuge (J2 MI, Beckman instruments Inc. Palo Alto, C.A. USA) using JA-17 rotor and thesupernatant was then recentrifuged at 35,000g for 30 min. The pellet was resus-pended in 300 mM mannitol and 5 mM Tris–HEPES, pH 7.4, with four passes by aloose fitting Dounce homogenizer (Wheaton, IL, USA) and centrifuged at 35,000gfor 20 min in a 15 ml corex tube. The outer white fluffy pellet of BBM was resus-pended in small volume of buffered 300 mM mannitol. Aliquots of homogenatesand BBM were saved and stored at �20 �C for BBM enzyme analysis. Each sampleof BBM was prepared by pooling tissues from two to three rats.

2.6. Serum/urine chemistries

Serum samples were deproteinated with 3% trichloroacetic acid in a ratio of 1:3,left for 10 min and then centrifuged at 2000g for 10 min. The protein free superna-tant was used to determine inorganic phosphate and creatinine. The precipitate wasused to quantitate total phospholipids. Blood urea nitrogen (BUN) and cholesterollevels were determined directly in serum samples. Glucose was estimated by o-tol-uidene method using kit from Span diagnostics, Mumbai, India. All these parame-ters were determined by standard procedures as mentioned in a previous study(Khundmiri et al., 2004).

Salineinjection (i.p) (0.9%)

ND

CPF

FO

(for 10 days) FO

C

CP

0 Cisplatin injection (i.p) 6mg/kgbodyweight

4 days

Blood

Kidney

Cortex

Medulla

Homogenate

Homogenate

BBMV

Sacrifice21th day

Sacrifice

Fig. 1. Experimental design (ND: normal diet; C: control; FO: fish oil diet; CP: cisplatin; CPF: fish oil + cisplatin; i.p.: intraperitoneal; CP injection ( ); normal saline injection( ).

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2.7. Assay of carbohydrate metabolism enzymes

The activities of the enzymes involving oxidation of NADH or reduction of NADPwere determined spectrophotometrically on Cintra 5 fixed for 340 nm using 3 ml ofassay buffer in a 1-cm cuvette at room temperature (28–30 �C). The enzyme activ-ities of lactate dehydrogenase (LDH, E.C.1.1.1.27), malate dehydrogenase (MDH,E.C.1.1.1.37), malic enzyme (ME, E.C.1.1.1.40), glucose-6-phosphate dehydrogenase(G6PDH, E.C.1.1.1.49), glucose-6-phosphatase (G6Pase, E.C.3.1.3.3) and fructose-1,6-bisphosphatase (FBPase, E.C.3.1.3.11) were assayed as described by Khundmiriet al. (2004). Hexokinase was estimated by the method of Crane and Sols (1953)and the remaining glucose was measured by method of Nelson-Somogyi (Nelson,1944).

2.8. Assay of brush border membrane marker enzymes and lysosomal marker enzyme

The activities of alkaline phosphatase (ALP), leucine amino peptidase (LAP), c-glutamyl transferase (GGTase) and acid phosphatase (ACPase) were determinedas described by Farooq et al. (2004).

2.9. Assay of enzymes involved in free radical scavenging

Superoxide dismutase (SOD, E.C.1.15.1.1) was assayed by the method of Markl-und and Marklund (1974). Catalase (CAT, E.C.1.11.1. 6) and glutathione peroxidase(GSH-Px) activities were determined by the method of Giri et al. (1996) and Floheand Gunzler (1984), respectively.

2.10. Lipid peroxidation and total-SH group estimation

Total SH groups were determined by the method of Sedlak and Lindsay (1968).Lipid peroxidation (LPO) by the method of Ohkawa et al. (1979).

2.11. Statistical analyses

All data are expressed as mean ± SEM for at least 4–5 different preparations.Statistical evaluation was conducted by one-way ANOVA using Origin 6.1 software.A probability level of p < 0.05 was selected as indicating statistical significance.Most of the changes between various groups were compared with control valuesfor better understanding and clarity.

3. Results

The present work was undertaken to study detailed mechanismof CP-induced nephrotoxicity and other deleterious effects and itspossible protection by feeding x-3 fatty acids enriched fish oil dietto the rats. To address our hypothesis, the effect of CP alone and incombination with fish oil (FO) was determined on nephrotoxicityparameters with serum biomarkers and enzymes of oxidativestress, brush border membrane and carbohydrate metabolism inrat kidney tissues.

3.1. Effect of dietary fish oil (FO) on CP induced nephrotoxicity inserum and urinary parameters

Results summarized in Tables 1 and 2 show the effect of CPalone and in combination with FO on blood and urine chemistry.

CP treatment to control rats resulted in significant increase inserum creatinine (Scr) and blood urea nitrogen (BUN), but decreasein cholesterol, phospholipids (PL), inorganic phosphate and glucose

compared to control rats. These changes were associated with pro-found phosphaturia, proteinuria and glucosuria accompanied bydecreased creatinine clearance. FO diet alone caused significant in-crease in serum glucose, Pi and creatinine clearance, decrease inBUN, urinary Pi and protein excretions.

Feeding of FO diet to CP administered (CPF) rats resulted in sig-nificant reversal of various CP elicited deleterious effects on serumand urine parameters. FO prevented CP induced increase of Scr,BUN and decrease of serum Pi, PLP and glucose. CP-induced phos-phaturia, proteinuria and glucosuria were absent in CPF comparedto CP rats.

3.2. Effect of dietary fish oil (FO) on CP induced alterations inbiomarker enzymes of BBM and lysosomes

To assess the structural integrity of certain organelles e.g., plas-ma membrane (BBM) and lysosomes, the effect of CP alone and incombination with FO diet was determined on biomarker enzymesof BBM and lysosomes in the homogenates of renal cortex and me-dulla and isolated BBM preparations from renal cortex.

3.2.1. Effect of CP alone and with FO diet on biomarkers of BBM andlysosomes in the homogenates

The activities of alkaline phosphatase (ALP), c-glutamyl trans-peptidase (GGTase), leucine aminopeptidase (LAP) and acidphosphatase were determined under different experimental condi-tions in the homogenates of renal cortex and medulla (Table 3). CPtreatment to control rats caused significant reduction in the spe-cific activities of ALP (�35.82%), GGTase (�36.73%) and LAP(�29.98%) in cortical homogenate. The feeding of FO diet prior toCP treatment prevented CP elicited decrease in BBM enzyme activ-ities. As can be seen from the data CP induced decrease in BBM en-zyme activities were similarly prevented by FO diet. However, theactivity of acid phosphatase (ACPase) was increased (+32.15%) byCP in cortical homogenates and FO diet was able to prevent the in-crease in enzyme activity in a similar manner (Table 3).

The activities of BBM enzymes similar to the cortex were alsolowered in the medulla by CP administration (Table 3). The con-sumption of FO in combination with CP treatment resulted in thereversal of CP induced decrease in ALP (�42.21%), GGTase(�37.10%) and LAP (�23.6%) in the medulla. The activity of ACPase(+35.45%) was increased by CP treatment. However, this increasewas prevented by FO feeding to CP treated rats.

3.2.2. Effect of CP and CP plus FO diet on BBM markers in isolatedBBMV

The effect of CP and FO on BBM marker enzymes was furtheranalyzed in BBMV preparations isolated from the renal cortex (Ta-ble 4). The data shows a similar activity pattern of BBM enzymes asobserved in cortical homogenates However, the magnitude of theeffect specifically in ALP activity was much more pronounced inBBMV than in cortical homogenates (Fig. 2). Activities of ALP

Table 1Effect of fish oil (FO) on serum parameters with/without CP treatment.

Group Creatinine (mg/dl) BUN (mg/dl) Cholesterol (mg/dl) Phospholipid (mg/dl) Phosphate (lmol/ml) Glucose (mg/dl)

Control 0.999 ± 0.046 14.40 ± 0.669 129.2 ± 1.96 114.71 ± 0.86 2.3 ± 0.047 73.22 ± 4.07CP 2.012 ± 0.139 (+101.40%)* 34.24 ± 3.30 (+137.7%)* 115.79 ± 1.29 (�10.379%)* 80.68 ± 0.38 (�29.66%) 1.92 ± 0.123 (�16.52%)* 19.16 ± 0.59 (�73.83%)*

CPF 1.23 ± 0.06 (+23%)� 18.25 ± 0.890 (+26.73%)*,� 88.40 ± 1.33 (�31.57%)*,� 98.51 ± 3.58 (�14.12%)*,� 2 ± 0.063 (�13.04%)* 75.74 ± 3.48 (+3.44%)*,�

FO 0.965 ± 0.046 (�3.40%) 11.79 ± 0.430 (�18.125%)* 108.47 ± 2.63 (�16.04%)* 99.5 ± 1.97 (�13.25%)* 2.51 ± 0.03 (+9.13%) 83.06 ± 5.32 (+13.435)

Results are mean ± SEM for five different preparations.CP: cisplatin treated; CPF: fish oil + cisplatin treated; FO: fish oil diet; BUN: blood urea nitrogen.Values in parentheses represent percent change from control.* Significantly different from control.� Significantly different from CP at p < 0.05 by one way ANOVA.

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(�54.28%), GGTase (�30.78%) and LAP (�21.76%) were profoundlydeclined by CP treatment as compared to control rats. Fish oil (FO)dietary supplementation similar to the effect in the homogenatesappeared to lower the severity of the CP treatment. CP-induced de-crease in BBM enzyme activities was significantly prevented bydietary FO.

3.3. Effect of fish oil (FO) on CP induced alterations in metabolicenzymes in renal cortex and medulla

The effect of CP, FO diet and their combined treatment wasdetermined on the activities of various enzymes of carbohydratemetabolism in renal cortex and medulla.

3.3.1. Effect on carbohydrate metabolism in renal cortexAs shown in Tables 5a and 6a, CP treatment to control rats sig-

nificantly increased the activity of lactate dehydrogenase (LDH)and hexokinase (HK) but decreased malate dehydrogenase(MDH), glucose-6-phosphatase (G6Pase) and fructose-1,6-bisphos-phatase (FBPase) activities in the renal cortex. When CP treatmentwas extended to FO fed rats, CP-induced alterations in metabolicenzyme activities were not only prevented by FO diet, but G6Paseactivity remained significantly higher in CPF compared to CPgroups.

The effect of FO, CP and their combined effect was also deter-mined on glucose-6-phosphate dehydrogenase (G6PDH) andNADP-malic enzyme (ME), source of NADPH needed in variousanabolic reactions (Table 6a). CP treatment to control rats signifi-cantly decreased G6PDH but increased ME activity. However, CP

Table 2Effect of fish oil (FO) on urine parameters with/without CP treatment.

Group Urine flow rate (UFR) (ml/day)

Creatinine clearance (ml/min/100 g bodyweight)

Phosphate (lmol/ml) Protein (mg/mmolcreatinine)

Glucose (mg/dl)

Control 18 ± 1.73 0.655 ± 0.002 1.16 ± 0.023 0.029 ± 0.003 13.98 ± 1.73CP 24 ± 0.57 (+33.33%)* 0.212 ± 0.057 (�67.63%)* 1.65 ± 0.77 (+42.24%)* 0.103 ± 0.004 (+255.17%)* 56.47 ± 5.51 (+303.93%)*

CPF 21 ± 2.88 (+16.66%) 0.355 ± 0 (�45.80%)* 1.19 ± 0.028 (+2.58%)� 0.064 ± 0.023 (+120.68%) 33.16 ± 1.69 (+137.19%)*,�

FO 20 ± 0.57 (+11.11%) 0.954 ± 0.0012 (+45.64%)* 0.644 ± 0 (�44.48%)* 0.020 ± 0 (�31.03%)* 4.66 ± 0.715 (�66.66%)*

Results are mean ± SEM for five different experiments.Values in parentheses represent percent change from control.CP: cisplatin treated; CPF: fish oil + cisplatin treated; FO: fish oil diet.* Significantly different from control.� Significantly different from CP at p < 0.05 by one way ANOVA.

Table 3Effect of fish oil (FO) on biomarkers of BBM and lysosomes in homogenates of (a) cortex and (b) medulla with/without CP treatment.

Enzyme ALP (lmol/mg protein/h) GGTase (lmol/mg protein/h) LAP (lmol/mg protein/h) ACPase (lmol/mg protein/h)Groups

(a) CortexControl 7.48 ± 0.54 10.18 ± 5.91 12.34 ± 1.45 3.39 ± 0.088CP 4.8 ± 0.215 (�35.82%)* 6.44 ± 1.29 (�36.73%) 8.64 ± 1.12 (�29.98%)* 4.48 ± 0.194 (+32.15%)*

CPF 5.50 ± 0.286 (�26.47%)*,� 6.92 ± 1.068 (�32.02%)� 11.07 ± 1.36 (�10.29%)� 3.52 ± 0.142 (+3.83%)�

FO 6.65 ± 0.504 (�11.09%) 12.07 ± 1 (+18.56%) 11.86 ± 2.23 (�3.89%) 3.03 ± 0.314 (�10.61%)

(b) MedullaControl 5.33 ± 0.331 12.91 ± 1.205 11.8 ± 1.14 2.51 ± 0.196CP 3.08 ± 0.246 (�42.21%)* 8.12 ± 0.552 (�37.10%)* 9.01 ± 0.690 (�23.64%)* 3.40 ± 0.096 (+35.45%)*

CPF 3.75 ± 0.849 (�29.64%)* 9.13 ± 1.33 (�29.28%)� 9.19 ± 0.503 (�22.11%)* 2.69 ± 0.177 (+7.17%)�

FO 4.49 ± 0.920 (�15.75%) 14.61 ± 2.66 (+13.17%) 9.79 ± 0.173 (�17.03%)* 2.53 ± 0.0914 (+0.796%)

Results (specific activity expressed as lmol/mg protein/h) are mean ± SEM for five different experiments.Values in parentheses represent percent change from control.CP: cisplatin treated; CPF: fish oil + cisplatin treated; FO: fish oil diet; ALP: alkaline phosphatase; GGTase: c-glutamyl transferase; LAP: leucine aminopeptidase; ACPase: acidphosphatase; BBM: brush border membrane.* Significantly different from control.� Significantly different from CP at p < 0.05 by one way ANOVA.

Table 4Effect of fish oil (FO) on activities of biomarker enzymes of BBM in cortical BBMV with/without CP treatment.

Enzyme ALP (lmol/mg protein/h) GGTase (lmol/mg protein/h) LAP (lmol/mg protein/h)Groups

Control 45.41 ± 5.53 81.22 ± 4.19 66.56 ± 2.5CP 20.76 ± 1.46 (�54.28%)* 56.28 ± 3.44 (�30.71%)* 52.07 ± 2.2 (�21.76%)CPF 23.76 ± 2.51 (�47.67%)* 78.1 ± 2.49 (�3.84%)*,� 66.17 ± 9.5 (�0.585%)�

FO 42.74 ± 1.52 (�5.87%) 94.15 ± 0.815 (+15.91%)* 75.38 ± 3.01 (+13.25%)

Results (specific activity expressed as lmol/mg protein/h) are mean ± SEM for five different experiments.Values in parentheses represent percent change from control.CP: cisplatin treated; CPF: fish oil + cisplatin treated; FO: fish oil diet; ALP: alkaline phosphatase; GGTase: c-glutamyl transferase; LAP: leucine aminopeptidase; BBM: brushborder membrane; BBMV: brush border membrane vesicles.* Significantly different from control.� Significantly different from CP at p < 0.05 by one way ANOVA.

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treatment to FO fed rats reduced the CP induced alterations in glu-cose-6-phosphate dehydrogenase (G6PDH) and NADP–malic en-zyme (ME) activities.

3.3.2. Effect on carbohydrate metabolism in renal medullaThe activities of LDH, MDH, HK, G6Pase and FBPase in medul-

lary homogenates were similarly affected by CP treatment (Tables5b and 6b) as in the cortical homogenates. Feeding of FO to CP trea-ted rats prevented CP induced alterations in various enzyme activ-ities. Similar to cortex, G6PDH activity decreased whereas MEactivity increased by CP treatment in the medulla (Table 6b). Die-tary supplementation of FO to CP treated rats decreased G6PDHand ME activity.

3.4. Effect of dietary fish oil (FO) on CP induced alterations inantioxidant defense parameters in renal cortex and medulla

It is evident that reactive oxygen species generated by varioustoxicants are important mediators of cellular injury and pathogen-esis of various diseases (Walker et al., 1999). Antioxidant status is apotential biomarker to determine the physiological state of the cell,tissue or organ. To ascertain the role of antioxidant system in CP-induced toxicity, the effect of CP was observed on oxidative stressparameters. CP enhanced lipid peroxidation (LPO) and significantlyaltered antioxidant enzymes both in cortex and medulla, albeit dif-ferently (Table 7). LPO measured in terms of malodialdehyde (MDAlevels was significantly enhanced in the cortex (+63.73%) and me-dulla (+28.9%), whereas total-SH declined in these tissue (�30.36%)and (�33.33%), respectively. CP treatment caused significant de-crease in superoxide dismutase (SOD, �45.69%), Glutathione per-oxidase (GSH-Px, �40.74%) and catalase (�19.96%) activities inthe renal cortex. In medulla, however the activity of SOD(�51.98%), catalase (�59.15%) and GSH-Px (�38.46%) were pro-foundly decreased by CP administration alone. However, FO con-sumption significantly enhanced antioxidant parameters. CPadministration to FO fed rats prevented the decline in superoxidedismutase (SOD), Glutathione peroxidase (GSH-Px) and catalase.The results indicate marked protection by FO diet against CP in-duced oxidative damage to renal tissues.

4. Discussion

Cisplatin is an effective chemotherapeutic agent that is widelyused in the treatment of many malignant tumors, including head

ALP

GGTase

Spec

ific

act

ivity

(m

mol

/mg

prot

ein/

h)Sp

ecif

ic a

ctiv

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ein/

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(a)

(b)

LAP

Spec

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act

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(mm

ol/m

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otei

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*

*†

†

(c)

Fig. 2. Effect of fish oil (FO) consumption on: (a) ALP; (b) GGTase; (c) LAP in cortexhomogenate and BBM with/without cisplatin (CP) treatment.

Table 5Effect of fish oil (FO) on activities of HK, LDH and MDH in homogenates of (a) cortex and (b) medulla with/without CP treatment.

Enzyme HK (lmol/mg protein/h) LDH (lmol/mg protein/h) MDH (lmol/mg protein/h)Groups

(a) CortexControl 1.48 ± 0.155 2.54 ± 0.32 2.38 ± 0.08CP 2.50 ± 0.069 (+68.91%)* 4.72 ± 0.08 (+85.82%)* 2.01 ± 0.04 (�15.54%)*

CPF 2.24 ± 0.194 (+51.35%) 4.11 ± 2.49 (+61.81%)� 2.36 ± 0.175 (�0.84%)FO 1.85 ± 0.005 (+25%) 3.50 ± 0.167 (+37.79%)* 3.34 ± 0.165 (+40.33%)*

(b) MedullaControl 5.32 ± 0.325 2.33 ± 0.255 13.18 ± 0.51CP 7.19 ± 0.325 (+35.15%)* 3.92 ± 0.466 (+68.24%) 8.79 ± 0.575 (�33.31%)*

CPF 4.4 ± 0.33 (�17.29%)� 3.76 ± 0.125 (+61.37%)* 8.84 ± 0.675 (�32.92%)*

FO 3 ± 0.641 (�43.60%)* 2.69 ± 0.115 (+15.45%) 10.88 ± 0.68 (�17.45%)*

Results (specific activity expressed as lmol/mg protein/h) are mean ± SEM for five different experiments.Values in parentheses represent percent change from control.CP: cisplatin treated; CPF: fish oil + cisplatin treated; FO: fish oil diet; HK: hexokinase; LDH: lactate dehydrogenase; MDH: malate dehydrogenase.* Significantly different from control.� Significantly different from CP at p < 0.05 by one way ANOVA.

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and neck, ovarian, and testicular cancers. However, the most com-mon adverse effect, nephrotoxicity, limits the use of this drug inmany cancer patients (Liu et al., 2006). Experimental evidencehas suggested that CP deteriorates renal function (Arany and Safir-stein, 2003) and glomerular filtration rate (GFR) in a dose-depen-dent manner (Yao et al., 2007). The alterations in the kidneyfunctions induced by cisplatin are closely associated with an in-crease in lipid peroxidation and reactive oxygen species (ROS) inrenal tissue (Baligha et al., 1998; Silici et al., 2010). In tumorsand other dividing cells, cisplatin–DNA crosslinks are thought tobe the cytotoxic lesion (Fink and Howell, 2000). Nonproliferatingcells are less sensitive to the toxicity of DNA-damaging agents,yet the quiescent proximal tubule cells are selectively killed by cis-platin (Lieberthal et al., 1996). The metabolic activation of CP inproximal tubular cells is found to be essential for its nephrotoxi-city. Both in vivo and cell culture studies provide evidence that cis-platin is metabolized to a nephrotoxin through a GSH-conjugateintermediate. GGTase is the first enzyme in the proposed metabolicpathway for activation of cisplatin glutathione conjugates to anephrotoxin (Mistry et al., 1991; Townsend et al., 2003). The ap-proach of identifying naturally occurring dietary sources and usingthem as cytoprotectants provides a strategy that is of great interestfor chemotherapy. In fact, a number of studies have evaluated a

wide range of antioxidants as potential renal cytoprotectors withpossible application as adjuvants to cisplatin chemotherapy. How-ever, most of them have not yet been proved safe for clinical appli-cation (Ali and Al Moundhri, 2006; Gamal el-Din et al., 2006).

The past three decades have been a period of rapid expansion inthe scientific knowledge of x-3 PUFAs. Recent studies have shownthat FO enriched in x-3 fatty acids retard the progression of vari-ous forms of cancers, depression, arthritis, asthma, cardiovascularand renal disorders (Caterina et al., 1994). However, studies onpossible beneficial effects of FO on drug or chemical induced neph-rotoxicity are very limited. Recently it has been documented thatFO protects against gentamicin-induced nephrotoxicity (Priyamva-da et al., 2008). The present studies were performed to test thehypothesis that FO consumption would ameliorate CP-inducednephrotoxic and other adverse effects thus maximizing the clinicaluse of CP in the treatment of various malignancies without any ma-jor side effects.

Single CP injection was found to cause marked renal dysfunc-tion as evident by increased serum creatinine and urea, diagnos-tic indicators of nephrotoxicity, accompanied by the significantdecrease in serum glucose, inorganic phosphate (Pi), phospholip-ids and cholesterol. The results show a typical pattern of CP-in-duced (Khan et al., 2009a,b) nephrotoxicity as indicated by

Table 6Effect of fish oil (FO) on activities of G6Pase, FBPase, G6PDH and ME in homogenates of (a) cortex and (b) medulla with/without CP treatment.

Enzyme G6Pase (lmol/mg protein/h) FBPase (lmol/mg protein/h) G6PDH (lmol/mg protein/h) ME (lmol/mg protein/h)Groups

(a) CortexControl 1.91 ± 0.121 3.44 ± 0.068 0.288 ± 0.0065 0.215 ± 0.009CP 1.536 ± 0.046 (�19.58%)* 2.05 ± 0.157 (�40.4%)* 0.195 ± 0.011 (�32.29%)* 0.266 ± 0.01 (+23.72%)*

CPF 1.83 ± 0.101 (�4.18%)� 2.26 ± 0.095 (�34.3%)* 0.223 ± 0.024 (�22.56%)* 0.245 ± 0.006 (+13.95%)FO 2.46 ± 0.037 (+28.79%) 2.60 ± 0.202 (�24.41%)* 0.293 ± 0.013 (+1.73%) 0.226 ± 0.009 (+5.11%)

(b) MedullaControl 2.83 ± 0.104 5.82 ± 0.635 0.922 ± 0.063 2.49 ± 0.085CP 2.73 ± 0.237 (�3.53%) 3.64 ± 0.301 (�37.45%)* 0.217 ± 0.032 (�76.46%)* 3.58 ± 0.037 (+43.77%)*

CPF 2.53 ± 0.127 (�10.6%) 3.76 ± 0.205 (�35.39%) 0.325 ± 0.015 (�64.75%)*,� 1.57 ± 0.275 (�36.94%)�

FO 2.05 ± 0.641 (�27.56%)* 5.61 ± 0.15 (�3.60%) 0.712 ± 0.06 (�22.77%)* 0.170 ± 0.0029 (�93.17%)

Results (specific activity expressed as lmol/mg protein/h) are mean ± SEM for five different experiments.Values in parentheses represent percent change from control.CP: cisplatin treated; CPF: fish oil + cisplatin treated; FO: fish oil diet; G6Pase: glucose-6-phosphatase; FBPase: fructose-1,6- bisphosphatase; G6PDH: glucose-6-phosphatasedehydrogenase; ME: malic enzyme.* Significantly different from control.� Significantly different from CP at p < 0.05 by one way ANOVA.

Table 7Effect of fish oil (FO) on enzymatic and non enzymatic antioxidant parameters in homogenates of (a) cortex and (b) medulla with/without CP treatment.

Parameters Lipid peroxidation(nmol/g tissue)

Total SH(lmol/g tissue)

SOD(lmol/mg protein)

Catalase(lmol/mg protein/min)

GSH-Px(lmols/mg protein/ min)

Groups

(a)CortexControl 94.61 ± 12.92 15.38 ± 0.125 25.67 ± 0.035 76.13 ± 1.01 0.0027 ± 0CP 154.91 ± 9.2 (+63.73%)* 10.71 ± 0.237 (�30.36%)* 13.94 ± 1.106 (�45.69%)* 60.93 ± 1.18 (�19.96%)* 0.0016 ± 0 (-40.74%)*

CPF 111.79 ± 5.42 (+18.15%)� 13.07 ± 0.751 (�15.01%)� 22.07 ± 0.905 (�14.02%)� 67.33 ± 144 (�11.55%)*,� 0.0018 ± 0(-33.33%)FO 102.11 ± 8.6 (+7.92) 15.03 ± 1.533 (�2.27%) 35.68 ± 2.26 (+38.99%)* 83.55 ± 1.17 (+9.74%)* 0.003 ± 3.84(+11.11%)

(b) MedullaControl 27.3 ± 1.72 1.47 ± 0.078 25.16 ± 1.73 97.66 ± 3.43 0.013 ± 9.27CP 35.19 ± 1.72 (+28.9%)* 0.98 ± 0.023 (�33.33%)* 12.08 ± 0.557 (�51.98%)* 39.89 ± 2.38 (�59.15%)* 0.008 ± 4.33*(-38.46%)CPF 33.60 ± 1.34 (+23.07%)* 0.105 ± 0.061 (�92.85%)� 23.82 ± 0.935 (�5.32%)� 56.09 ± 2.76 (�42.56%)*,� 0.0093 ± 4.48*�(-28.46%)FO 31.27 ± 0.25 (+14.54%) 1.29 ± 0.005 (�12.24%) 34.62 ± 1.19 (+37.59%)* 111.69 ± 0.715 (+14.36%)* 0.015 ± 0.002*(+15.38%)

Results are mean ± SEM for five different experiments.Values in parentheses represent percent change from control.CP: cisplatin treated; CPF: fish oil + cisplatin treated; FO: fish oil diet; LPO: lipid peroxidation; total-SH: sulphydryl groups; SOD: superoxide dismutase; GSH-Px: glutathioneperoxidase.* Significantly different from control.� Significantly different from CP at p < 0.05 by one way ANOVA.

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massive proteinuria, glucosuria and phosphaturia. The feeding ofFO prior to and after CP treatment significantly lowered CP-elic-ited increased levels of both serum creatinine and BUN. Serumglucose and phospholipids were improved upon CP treatmentto FO consuming rats.

Since the BBM of renal proximal tubules has been shown asthe major site of CP-induced renal injury (Arany and Safirstein,2003; Fatima et al., 2004), the integrity of the membrane wasassessed by the status of its biomarker enzymes ALP, GGTaseand LAP. CP caused significant decrease in the activities of ALP,GGTase and LAP in the cortical homogenates as well as in iso-lated BBM vesicles. A similar decrease was observed in medulla,suggesting an overall CP induced damage to the kidney. The de-crease in BBM enzyme activities might have occurred due to lossof BBM enzymes and other proteinic components from damagedBBM into the lumen that later appear in urine as reported earlier(Khan et al., 2009a,b; Banday et al., 2008; Fatima et al., 2004). Incontrast to CP, FO consumption, however, enhanced the activitiesof BBM enzymes in homogenate and BBM, indicating an overallimprovement in renal BBM integrity. FO consumption in combi-nation with CP treatment prevented/retarded CP-induced de-crease of BBM enzyme activities in the renal tissues. CP isshown to be metabolized to a nephrotoxin by renal proximal tu-bule cells and the BBM marker enzyme, GGTase plays an essen-tial role in the pathway involved (Townsend et al., 2003).Inhibition of GGtase activity has been shown to block CP toxicity(Townsend et al., 2003; Townsend and Hanigan, 2002). We spec-ulate that FO might be inhibiting the activity of an essential en-zyme in this pathway thereby preventing formation ofnephrotoxin and hence reducing CP nephrotoxicity. The activityof GGTase was measured in all the experimental groups. GGTaseactivity was found to increase in FO fed rats while it decreasedin CP treated rats as compared to control suggesting that FOmight be ameliorating the effect of CP by inhibiting another en-zyme and not GGTase crucial/essential for the pathway involvedin metabolic activation of CP to a nephrotoxin. The activity oflysosomal enzyme, ACPase was significantly increased in the cor-tex and medulla by CP treatment. Alteration in ACPase activitydemonstrates CP-induced loss of lysosomal function (Kuhlmannet al., 1997; Courjault-Gautier et al., 1995).

Since the major function of kidney is to reabsorb importantions and molecules, that inturn depends on the structural integ-rity of BBM and available energy as ATP which is supplied byvarious metabolic pathways, it is imperative that any alterationsto these metabolic pathways caused by toxic insult or otherwisemodified would affect the functional capabilities of kidney. Toassess the functional aspects, the activities of various enzymesof carbohydrate metabolism were examined under differentexperimental conditions. The activities of various enzymes in-volved in glycolysis, TCA cycle, gluconeogenesis and HMP-shuntpathway were differentially altered by CP treatment and/or byFO consumption. CP caused significant increase in LDH and de-crease in MDH in the renal cortex and medulla, which was asso-ciated with simultaneous increase in hexokinase activity in therenal tissues. Although the actual rates of glycolysis or TCA cyclewere not determined, marked decrease in MDH activity appearsto be due to CP-induced damage to mitochondria (Kuhlmannet al., 1997; Zhang and Lindup, 1993). A marked increase inLDH and to some extent hexokinase activity with simultaneousdecline in TCA cycle enzyme, MDH appears to be an adaptivecellular effect in energy metabolism from aerobic metabolismalternatively to anaerobic glycolysis due to CP induced mito-chondrial dysfunction.

CP also differentially altered the activities of enzymes of gluco-neogenesis and HMP-shunt pathway. The activities of G6Pase andFBPase (gluconeogenesis) and G6PDH (HMP shunt), however,

profoundly decreased in both cortex and medulla albeit to differentextent. However, the activity of NADP malic enzyme (ME) variablyincreased in the renal tissues. The present data indicate that CPcaused differential effect on different enzymes of the carbohydratemetabolism. FO administration to CP-treated rats resulted in over-all improvement of carbohydrate metabolism as evident by higheractivities of LDH, MDH and gluconeogenic enzymes in CPF com-pared to CP alone or control rats. This could be attributed to thefact that FO might have lowered the number of damaged mito-chondria or affected macromolecules or increased the number ofnormally active organelles or macromolecules.

Earlier reports reveal that heavy metals including CP exert theirtoxic effects by inducing the generation of reactive oxygen species(ROS) (Fatima et al., 2004; Baligha et al., 1998; Banday et al., 2008).A major cellular defense against ROS is provided by SOD and cata-lase, which together convert superoxide radicals first to H2O2 andthen to molecular oxygen and water. Other enzymes e.g., GSH-Pxuse thiol-reducing power of glutathione to reduce oxidized lipidsand protein targets of ROS. CP has been shown to enhance lipidperoxidation (LPO), an indicator of tissue injury and deplete GSHand protein thiols (Mistry et al., 1991). The present result showthat CP administration to control rats caused severe damage to re-nal tissues most likely by ROS generation as apparent by perturba-tion in the antioxidant enzymes (SOD, Catalase and GPx-SH) andtotal-SH content that lead to increased lipid peroxidation. Theseresults were consistent with earlier reports that oxidative stresswas thought to contribute to the pathogenesis of the tissue inwhich the mechanism of nephrotoxicity in cisplatin-treated miceinvolved the generation of free radicals and reactive oxygen spe-cies (ROS) (Atessahin et al., 2005; Sueishi et al., 2002) and causingdepletion of enzymatic and nonenzymatic antioxidants (Ajith et al.,2007). The feeding of FO diet to CP treated rats prevented CP in-duced augmentation of LPO and suppression of antioxidant en-zyme activities. The protection against CP effect by FO can beattributed to its intrinsic biochemical and natural antioxidantproperties. Thus, it appears that FO enriched in x-3 fatty acids en-hanced resistance to free radical attack generated by CP adminis-tration similarly as demonstrated in lupus nephritis and otherpathologies (Chandraseka and Fernandes, 1994; Xi and Chen,2000; Donadio, 2001).

Although the mechanism involved in cisplatin nephrotoxicityhave been extensively studied, they are not yet fully elucidated.It has been shown that CP induces necrosis of the S3 subseg-ment of proximal tubule (Townsend et al., 2003; Portilla et al.,2006). CP has been shown to be activated to a potent nephro-toxin in renal proximal tubules (Townsend et al., 2003). CPnephrotoxicity has been morphologically characterized by lossof microvilli, alteration in the number and size of lysosomesand mitochondrial vacuolization accompanied by functional dis-turbances of various cell organelles (Kuhlmann et al., 1997). Dif-ferent studies have shown that cytotoxicity of CP is probably dueto combination of insults including peroxidation of cell mem-brane, mitochondrial dysfunction, inhibition of protein synthesisand DNA damage in the kidney (Santos et al., 2007). It has beensuggested that CP induces renal damage by free radical genera-tion, such as hydroxyl radical and superoxide anion, by alteringarginine metabolism, by increasing activity of calcium indepen-dent nitric oxide synthase and more recently by apoptosis (San-tos et al., 2007; Devipriya and Shyamaladevi, 1999; Arany et al.,2004). However, CP-induced oxidative stress was shown to bestrongly involved in acute renal damage (Gonzales et al.,2005). The present findings at least in part support above pro-posed mechanism. To summarize, our results (Fig. 3) indicatean overall improvement in metabolic activities, BBM integrityand antioxidant defenses upon dietary FO supplementation toCP treated rats.

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5. Conclusion

The present results show that CP elicited deleterious nephro-toxic and other adverse effects as indicated by significant increasein serum creatinine, BUN, decrease in the activities of various en-zymes of BBM, mitochondria and metabolism. The effects appearedto be mediated, at least, in part due to CP-induced perturbation inantioxidant defense mechanism. FO consumption to a larger extentprevented CP-induced nephrotoxicity parameters by enhancingenergy metabolism, BBM enzymes and by strengthening antioxi-dant defense mechanism. Taking into account these results, wepropose that FO may maximize the clinical use of CP in the treat-ment of various malignancies without nephrotoxic and other sideeffects.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

Indian Council of Medical Research (ICMR), New Delhi, India isacknowledged for the award of JRF (Junior Research Fellowship)/SRF (Senior Research Fellowship) to W.K. University Grants Com-mission (UGC) for the award of scholarship to A.N. and S.R. andfinancial support to the department from University Grants Com-mission (UGC-DRF) is also gratefully acknowledged.

References

Abdel-Gayoum, A.A., Bashir, A.A., El-Fakhri, M.M., 1995. Effect of fish oil andsunflower oil supplementations on gentamicin induced nephrotoxicity in rat.Hum. Exp. Toxicol. 14, 884–888.

Ajith, T.A., Usha, S., Nivitha, V., 2007. Ascorbic acid and alphatocopherol protectanticancer drug cisplatin induced nephrotoxicity in mice: a comparative study.Clin. Chim. Acta 375, 82–86.

Ali, B.H., Al Moundhri, S.M., 2006. Agents ameliorating or augmenting thenephrotoxicity of cisplatin and other platinum compounds: a review of somerecent research. Food Chem. Toxicol. 44, 1173–1183.

Antonio, Z., Cafaggi, S., Mariggio, M.A., Vannozzi, M.O., Bocchini, M.V., Caviglioli, G.,Viale, M., 2002. Reduction of cisplatin hepatotoxicity by procainamidehydrochloride in rats. Eur. J. Pharmacol. 442, 265–272.

Arany, I., Safirstein, R., 2003. Cisplatin nephrotoxicity. Semin. Nephrol. 23, 460–464.Arany, I., Megyesi, J.K., Kaneto, H., Price, P.M., Safirstein, R.L., 2004. Cisplatin-

induced cell death is EGFR/src/ERK signaling dependent in mouse proximaltubule cells. Am. J. Physiol. Renal Physiol. 287, 543–549.

Aronson, W.J., Glaspy, J.A., Reddy, S.T., Reese, D., Heber, D., Bagga, D., 2001.Modulation of n-3 and n-6 polyunsaturated fatty acid ratios with dietary fishoils in men with prostrate cancer. Urology 58, 283–288.

Atessahin, A., Yilmaz, S., Karahan, I., Ceribasi, A.O., Karaoglu, A., 2005. Effects oflycopene against cisplatin-induced nephrotoxicity and oxidative stress in rats.Toxicology 212, 116–123.

Baligha, R., Zhang, Z., Baliga, M., Ueda, N., Shah, S.V., 1998. Invitro and invivoevidence suggesting a role of iron in cisplatin-induced nephrotoxicity. KidneyInt. 53, 394–401.

Banday, A.A., Farooq, N., Priyamvada, S., Yusufi, A.N.K., Khan, F., 2008. Effect ofuranyl nitrate on enzymes of carbohydrate metabolism and brush bordermembrane in different kidney tissues. Food Chem. Toxicol. 46, 2008.

Caterina, R.D., Endres, S., Kristensen, S.D., Schimdt, E.B., 1994. N-3 fatty acids andrenal diseases. Am. J. Kidney. Dis., 397–415.

Chandraseka, B., Fernandes, G., 1994. Decreased pro-inflammatory cytokines andincreased antioxidant enzyme gene expression by x-3 lipids in murine lupusnephritis. Biochem. Biophys. Res. Commun. 200, 893–898.

Courjault-Gautier, F., Le Grimellec, C., Giocondi, M.C., Toutain, H.J., 1995.Modulation of sodium coupled uptake and membrane fluidity by cisplatin inrenal proximal tubular cells in primary culture and BBM vesicles. Kidney Int. 47,1048–1056.

Crane, R.K., Sols, A., 1953. The association of particulate fractions of brain and othertissue homogenates. J. Biol. Chem. 203, 273–292.

Devipriya, S., Shyamaladevi, C.S., 1999. Protective effect of quercetin in cisplatininduced cell injury in the rat kidney. Indian J. Pharmacol. 31, 422–426.

Donadio, J.V., 2001. The emerging role of omega-3 polyunsaturated fatty acids in themanagement of patients with IgA nephropathy. J. Ren. Nutr. 11, 122–128.

Farooq, N., Yusufi, A.N.K., Mahmood, R., 2004. Effect of fasting on enzymes ofcarbohydrate metabolism and brush border membrane in rat intestine. Nutr.Res. 24, 407–416.

CP

CP+ FO

Oxidative stressIncrease in LPO, Decrease

in antioxidant enzymes

Decrease in lysosomal integrity

Mitochondrial dysfunctionLoss of TCA cycle enzyme

Damage to proximal tubular membrane

Effacement of BBMV Decrease in AlkPase; GGTase;

LAP;

Increased lysosomal integrity

Improved mitochondrial functioning

Reduced damage to proximal tubular

Diminished Oxidative stress

Fig. 3. Cisplatin (CP) induced alterations in cell metabolism, membrane integrity and oxidative stress and its amelioration by dietary fish oil (FO): a summary.

272 A. Naqshbandi et al. / Food and Chemical Toxicology 50 (2012) 265–273

Author's personal copy

Fatima, S., Yusufi, A.N.K., Mahmood, R., 2004. Effect of cisplatin on renal brushborder membrane enzymes and phosphate transport. Hum. Exp. Toxicol. 23,547–554.

Fink, D., Howell, S.B., 2000. How does cisplatin kill cells? In: Kelland, L.R., Favilli, F.(Eds.), Platinum Based Drugs in Cancer Therapy. Humana Press, Totowa, NewJersey, pp. 149–167.

Flohe, L., Gunzler, W., 1984. Assays of glutathione peroxidase. In: Colowick, S.P.,Kaplan, N.O. (Eds.), Methods Enzymology. Academic Press, New York, pp. 114–121.

Gamal el-Din, A.M., Al-Bekairi, A.M., Carvedilol, 2006. A beta adrenoceptor blockerwith antioxidative potential, attenuates cisplatin-induced nephrotoxicity inrats. J. Appl. Sci. Res. 2, 331–335.

Giri, U., Iqbal, M., Athar, M., 1996. Porphyrin mediated photosensitization has aweak tumor promoting activity in mouse skin: possible role of in-situ generatedreactive oxygen species. Carcinogenesis 17, 2023–2028.

Gonzales, R., Romay, C., Borrego, A., Hernandez, F., Zamora, Z., Rojas, E., 2005. Lipidperoxides and antioxidant enzymes in cisplatin chronic nephrotoxicity in rats.Mediators Inflamm. 3, 139–143.

Hibbeln, J., 1998. Fish consumption and major depression. The Lancet 351,1213.

Hong, K.O., Hwang, J.K., Park, K.K., Kim, S.H., 2005. Phosphorylation of c-Jun N-terminal kinases (JNKs) is involved in the preventive effect of xanthorrhizol oncisplatin-induced hepatotoxicity. Arch. Toxicol. 79, 231–236.

Khan, S.A., Priyamvada, S., Khan, W., Khan, S., Farooq, N., Yusufi, A.N.K., 2009a.Studies on the protective effect of green tea extract against cisplatin inducednephrotoxicity. Pharma. Res. 60, 382–391.

Khan, S.A., Priyamvada, S., Farooq, N., Khan, S., Yusufi, A.N.K., 2009b. Influence ofgreen tea extract on gentamicin-induced nephrotoxicity and oxidative damagein rat kidney. Pharma. Res. 59, 254–262.

Khundmiri, S.J., Asghar, M., Khan, F., Salim, S., Yusufi, A.N.K., 2004. Effect of ischemiaand reperfusion on enzymes of carbohydrate metabolism in rat kidney. J.Nephrol. 17, 1–7.

Khundmiri, S.J., Asghar, M., Banday, A.A., Khan, F., Salim, S., Levi, M., Yusufi, A.N.K.,2005. Effect of reperfusion on sodium dependent phosphate transport in renalbrush border membranes. Biochim. Biophys. Acta 1716, 19–28.

Kuhad, A., Pikhwal, S., Sharma, S., Turkey, N., Chopra, K., 2007. Effect of curcumin ininflammation and oxidative stress in cisplatin-induced nephrotoxicity. FoodAgric. Chem. 55, 10150–10155.

Kuhlmann, M.K., Burkhardt, G., Kohler, H., 1997. Insights into potential cellularmechanisms of cisplatin nephrotoxicity and their clinical application. Nephrol.Dial. Transplant. 12, 2478–2480.

Lebwohl, D., Canetta, R., 1998. Clinical development of platinum complexes incancer therapy: an historical perspective and an update. Eur. J. Cancer 34, 1522–1534.

Leibbrandt, M.E.I., Grushenka, H.I.W., Metz, A.L., Ozobia, A.A., Haskins, J.R., 1995.Critical subcellular targets of cisplatin and related platinum analogs in rat renalproximal tubule cells. Kidney Int. 8, 761–770.

Lieberthal, W., Triaca, V., Levine, J., 1996. Mechanisms of death induced by cisplatinin proximal tubular epithelial cells: apoptosis vs. necrosis. Am. J. Physiol. 270,F700–F708.

Liu, M., Chien, C., Burne-Taney, M., Molls, R.R., Racusen, L.C., Colvin, R.B., Rabb, H.,2006. A pathophysiologic role for T-lymphocytes in murine acute cisplatinnephrotoxicity. J. Am. Soc. Nephrol. 17, 765–774.

Marklund, S., Marklund, G., 1974. Involvement of the superoxide anion radical inthe auto oxidation of pyrogallol and a convenient assay for superoxidedismutase. Eur. J. Biochem. 47, 469–474.

Matsushima, H., Yonemura, K., Ohishi, K., Hishida, A., 1998. The role of oxygen freeradicals in cisplatin induced acute renal failure in rats. J. Lab. Clin. Med. 12, 900–908.

Mistry, P., Merazza, Y., Spargo, D.J., Riley, P.A., Mcbrien, D.C.H., 1991. The effects ofcisplatin on the concentration of protein thiols and glutathione in the ratkidney. Cancer Chemother. Pharmacol. 28, 501–504.

Nelson, N.A., 1944. Photometric adaptation of the Somogyi method for thedetermination of glucose. J. Biol. Chem. 153, 375–381.

Nowak, G., 2002. Protein kinase C-alpha and ERK1/2 mediate mitochondrialdysfunction, decreases in active Na+ transport, and cisplatin-inducedapoptosis in renal cells. J. Biol. Chem. 27, 43377–43388.

Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues bythiobarbituric acid reaction. Anal. Biochem. 95, 351–358.

Portilla, D., Li, S., Nagothu, K.K., Megyesi, J., Kaissling, B., Schnackenberg, I., 2006.Metabolomic study of cisplatin induced nephrotoxicity. Kidney Int. 69, 2194–2204.

Posta, T.L., Gebauer, S.K., Kris-Etherton, P., 2006. Dietary omega-3 fatty acid intakeand cardiovascular risk. Am. J. Cardiol. 98, 3i–18i.

Priyamvada, S., Priyadarshini, M., Arivarasu, N.A., Farooq, N., Khan, S., Khan, S.A.,Khan, W.Md., Yusufi, A.N.K., 2008. Studies on the protective effect of dietary fishoil on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney.Prostaglandins Leukot. Essent. Fatty Acids 78, 369–381.

Ramesh, G., Reeves, W.B., 2002. TNF-alpha mediates chemokine and cytokineexpression and renal injury in cisplatin nephrotoxicity. J. Clin. Invest. 110, 835–842.

Santos, N.A., Catao, C.S., Martins, N.M., Curti, C., Bianchi, M.L., Santos, A.C., 2007.Cisplatin induced nephrotoxicity is associated with oxidative stress, redox stateunbalance, impairment of energetic metabolism and apoptosis in rat kidneymitochondria. Arch. Toxicol. 81, 495–504.

Sedlak, J., Lindsay, R.H., 1968. Estimation of total protein bound and non proteinbound SH groups in tissue with Ellman’s reagent. Anal. Biochem. 25, 192–205.

Silici, S., Ekmekcioglu, O., Kanbur, M., Deniz, K., 2010. The protective effect of royaljelly against cisplatin-induced renal oxidative stress in rats. World J. Urol. 29,127–132.

Simopoulos, A.P., 1991. Omega-3-fatty acids in health and disease and in growthand development. Am. J. Clin. Nutr. 54, 438–463.

Sueishi, K., Mishima, K., Makino, K., Itoh, Y., Tsuruya, K., Hirakata, H., 2002. Protectionby a radical scavenger edaravone against cisplatin-induced nephrotoxicity inrats. Eur. J. Pharmacol. 451, 203–208.

Thakkar, R.R., Wang, O.L., Zerouga, M., Stillwell, W., Haq, A., Kissling, R., 2000.Docosahexaenoic acid reverses cyclosporin-A induced changes in membranestructure and function. Biochim. Biophy. Acta 1474, 183–195.

Townsend, D.M., Hanigan, M.H., 2002. Inhibition of gamma-glutamyltranspeptidase or cysteine S-conjugate beta-lyase activity blocks thenephrotoxicity of cisplatin in mice. J. Pharmacol. Exp. Ther. 300, 142–148.

Townsend, D.M., Deng, M., Zhang, L., Lapus, M.G., Hangian, M.H., 2003. Metabolism ofcisplatin to a nephrotoxin in proximal tubule cells. J. Am. Soc. Nephrol. 14, 1–10.

Walker, P.D., Barri, Y., Shah, S.V., 1999. Oxidant mechanisms on gentamicinnephrotoxicity. Ren. Fail. 21, 433–442.

Xi, S., Chen, L.H., 2000. Effects of dietary fish oil on tissue glutathione and antioxidantdefense enzymes in mice with murine AIDS. Nutr. Res. 20, 1287–1299.

Xiao, T., Choudhary, S., Zhang, W., Ansari, N.H., Salahudeen, A., 2003. Possibleinvolvement of oxidative stress in cisplatin-induced apoptosis in LLC-PK1 cells.J. Toxicol. Environ. Health A 66, 469–479.

Yam, D., Peled, A., Shinitzy, M., 2001. Suppression of tumor growth and metastasisby dietary fish oil combined with vitamin E and C and cisplatin. CancerChemother. Pharmacol. 47, 34–40.

Yao, X., Panichpisal, K., Kurtzman, N., Nugent, K., 2007. Cisplatin nephrotoxicity: areview. Am. J. Med. Sci. 33, 115–124.

Zhang, J.G., Lindup, W.E., 1993. Role of mitochondria in cisplatin-induced oxidativedamage exhibited by rat renal cortical slices. Biochem. Pharmacol. 45, 2215–2222.

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